Project Summary The Molecular Systems Pathology Core will provide expert morphological and molecular evaluation of normal and tumor samples of human and experimental models to investigators of this Program Project. It will investigate the potential clinical relevance of pathways and lead compounds identified from the preclinical models in the projects. The Core will assist in the generation and validation of data regarding genetic alterations associated with tumorigenesis and temporal maps of early versus late tumor progression. The Core will support the overall goals of the Program through the application of novel technologies in the areas of object-oriented image analysis and quantitative biomarker multiplexing, allowing for integration of phenotype- genotype, and providing patient-specific risk profiles. It will also participate in the integration of data between Projects, and especially between Core B (Levine) and our Core C. The Core has ample tissue resources and will assist all Projects. These include well-characterized cohorts of human hepatocellular carcinomas (HCC), breast carcinomas, lymphoma and leukemia cases, plus multiple tissue microarrays, all cases linked to outcome data. Tissue specimens will be provided for laboratory studies under strict guidelines. The Specific Aims are: (1) To confirm histopathology and morphometric parameters of human tumors and murine models developed by all Projects. More specifically, well-characterized HCC cases will be use for the assessment of oncogenic gain of function of certain p53 mutants [Prives (Project 1) and Lowe (Project 4)]. (2) To develop and perform quantitative phenotypic assessment of in situ signaling pathways in the primary patient specimens and mouse models using multiplex immunofluorescence/multiplex immunohistochemistry combined with image analysis [e.g., Stockwell (Project 2) and Prives (Project 1) to determine and validate expression levels of biomarkers of ferroptosis and p53-regulated genes in mevalonic acid (MVA) pathway]. (3) To further enhance and conduct qualitative and quantitative microscopy assessment of heterochromatic marks to assist in the analysis of oncogene-induced chromatin transitions and DNA damage response (DDR) [e.g., Petrini (Project 3) analyses of pre- and post-oncogenic stresses with high resolution assays, such as ?H2AX, that allows quantification of foci in nuclei of cells of interest). (4) To characterize context dependencies of wild-type and mutant p53 effector programs, and p53-mediated gen repression in tumorigenesis and tumor maintenance [e.g., Lowe (Project4) pre- and post-treated analyses of transgenic mice with B cell lymphomas). (5) To explore the relationship between p53 activities, mutational loss and cancer stem cells from certain solid tumors; and assist in the analysis of p53 in regulating Ras-induced p53 senescence (Lowe and Levin).